The proposed study will focus on matrix vesicles (MVs) which play an initiating role in the mineralization of teeth and bones. MVs are submicroscopic, extracellular, membrane-invested particles that serve as the initial site of calcification in dentin, growth cartilage and developing bone. Our lab was involved in the first identification, isolation and characterization of MVs. We and others have provided evidence that MV phosphatases, including alkaline phosphatase (ALP), are involved in MV mineralization. Furthermore, these phosphatases are integrated into the MV membrane under which mineral first appears suggesting a critical role for ALP and other components of the MV membrane in initiating calcification. Our specific aims are directed toward an increased understanding of the molecular and structural organization of the MV membrane and sap with emphasis on identifying and localizing major constitutive proteins, testing the function of MV phosphatases in the calcification mechanism of isolated MVs, and studying the regulation of MV biogenesis by cultured bone cells and marrow osteoprogenitor cells. Proposed new studies include: 1) further analysis of the composition of isolated MVs to complete the identification of major proteins; 2) assignment of major MV constituents to the membrane versus the vesicle sap using high resolution EM immunolocalization coupled with biochemical release of membrane-attached proteins by surface active agents versus total release by detergents; 3) further characterization of the role of MV phosphatases in the mineralization of MVs from whole cartilage versus MVs generated in cultures of bone cells or marrow osteoprogenitor cells. Agents such as 1,250H2D3, dexamethasone, or fluoride, known to boost MV ALP activity, will be tested for a positive effect on MV calcification; 4) further studies will characterize the mechanism of MV biogenesis by cultured rat chondrocytes, osteoblasts and osteoprogenitor cells of the marrow stroma as modified by agents known to promote osseous differentiation in vitro including glucocorticoids, fluoride and retinoic acid. This is a fundamental study of the mechanism by which dental and skeletal forms of mineralization are initiated. New knowledge of the matrix vesicle calcification can be applied to a broad range of topics including specific diseases in which abnormal calcification occurs.